Cryomicroscopic analysis of freezing in liver of the freeze-tolerant wood frog

The technique of directional solidification coupled with low-temperature scanning electron microscopy was applied to analyze the freezing of liver slices from the freeze-tolerant frog Rana sylvatica. Micrographs of liver slices from 5°C-acclimated frogs frozen on the directional stage to -7°C showed continuous ice formed along an expanded vasculature with hepatocytes that were shrunken and virtually dehydrated. However, when frogs were given a survivable freezing exposure at -4°C for 24 h, liver slices subsequently frozen in vitro at -7°C were much less shrunken and the presence of intracellular ice crystals (formed when samples were plunged into liquid N₂ before microscopy) demonstrated that ample free water remained in these hepatocytes at -7°C. This reduced level of cell dehydration was correlated with the buildup of 280 ± 61 μmol/g wet wt glucose as a cryoprotectant in liver during the -4°C exposure in vivo. The study provides the first direct cytological analysis of the freezing process in an organ of a freeze-tolerant vertebrate and the first confirmation of the relationship between maintenance of a critical minimum cell volume and freezing survival by these animals.